The C2 Domain and Altered ATP-Binding Loop Phosphorylation at Ser³⁵⁹ Mediate the Redox-Dependent Increase in Protein...

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Signaling

Background:

• Protein kinase C-delta (PKCδ) plays diverse roles in cellular processes, with its function varying based on stimulus-specific signaling.
• PKCδ exhibits membrane-delimited actions upon agonist stimulation and altered activity during oxidative stress, releasing as a Tyr(313)-phosphorylated enzyme.

Purpose of the Study:

• To identify the structural mechanisms controlling PKCδ's altered activity during oxidative stress.
• To elucidate the interaction between the phosphorylated hinge region and the C2 domain of PKCδ.

Main Methods:

• Investigated the interaction between the Tyr(313)-phosphorylated hinge region and the phosphotyrosine-binding C2 domain of PKCδ.
• Utilized site-directed mutagenesis (e.g., S359A) to assess the impact on PKCδ activity and substrate specificity.
• Examined native PKCδ phosphorylation in cardiomyocytes under oxidative stress conditions.

Main Results:

• Identified a novel interaction between the Tyr(313)-phosphorylated hinge region and the C2 domain, which indirectly decreases Ser(359) phosphorylation in the kinase domain ATP-loop.
• Wild-type PKCδ with Ser(359) substitutions showed a preference for serine phosphoacceptors, while PKCδ-S359A exhibited lipid-independent activity towards both serine and threonine phosphoacceptors.
• Oxidative stress reduced Ser(359) phosphorylation in native cardiomyocytes, and PKCδ-S359A overexpression elevated basal phosphorylation of substrates with serine and threonine phosphoacceptors.

Conclusions:

• A C2 domain-pTyr(313) docking interaction dynamically regulates PKCδ catalytic activity by controlling ATP-positioning loop phosphorylation.
• This interaction represents a novel, physiologically relevant structural determinant of PKCδ enzymology, particularly under conditions like oxidative stress.